Project Summary My aim is to become an independent investigator leading a cutting-edge lab, in a leading microbiology department or medical school, and to become a leader in the field of Staphylococcal disease and antibiotic tolerance of persister cells. To further my progress toward this goal, I am proposing a multifaceted approach to determine the mechanism of persister formation under biologically relevant conditions and identify novel compounds to kill tolerant cells. I possess the skills, background and training in the key areas for this application. I have carefully designed this proposal and have recruited world class collaborators and consultants to facilitate the accomplishment of the project goals during the period of award and their progression beyond. This award will provide critical support and will facilitate my securing of a position in a leading research institute. The award will provide invaluable financial support in my early career as an independent investigator and will allow me to focus solely on research output for the duration of the award. Furthermore, the K22 will allow me to acquire skills that will aid in my progress as a unique interdisciplinary independent investigator and will allow me to achieve full separation from my current supervisor. Specifically, I will learn how to; 1) run Tn- seq experiments, 2) perform screens with P. aeruginosa to investigate how microbial interaction influences persisters 3) examine antibiotic tolerance in the intracellular environment 4) translate findings to drug-discovery pipelines and establish screens for anti-persister antibiotics. I will apply these skills, along with my current repertoire of capabilities to establish my independence and produce more ground-breaking research in the field of Staphylococcus chronic infection and novel drug therapies. I have created a network of exceptional scientists. Their hugely diverse skillsets and dedication to continued collaboration will allow me to produce multi-disciplinary research projects and will continue to be an invaluable learning experience. The diversification of skills that I develop while managing these collaborations will contribute to my development as an independent investigator with a unique insight and skillset. In this proposal, I will examine antibiotic tolerance under relevant conditions, elucidating the factors determining antibiotic tolerance in a multi-species environment and the intracellular environment. I have shown that under regular growth conditions, persisters are essentially equivalent to stationary phase cells and antibiotic tolerance of these cells is due to a decrease in ATP. I have also identified regulators regulators rex and mgrA and the clpP peptidase that control persister formation. I have developed a fluorescence based system for sorting antibiotic tolerant persisters from a heterogeneous population. I have also identified and optimized the acyldepsipeptides as anti-persister antibiotics due to their activation of ClpP and independence of ATP. I have also shown that co-culture with P. aeruginosa induces antibiotic tolerance in S. aureus. I hypothesize that the susceptibility to bactericidal action of antibiotics is determined by ATP levels, which are dependent on intracellular metabolic pathway activity, and extracellular stimuli encountered during infection, and that killing low ATP persisters will improve outcome of infection. This hypothesis will be tested by the following 3 aims: 1. Determine the mechanism of S. aureus persister induction by P. aeruginosa. 2. Determine the mechanism of S. aureus persister formation in the intracellular environment. 3. Identify novel inhibitors of persister formation. This is a multi-faceted, multi-disciplinary project based on the further development of high impact work. The project is realistic and comprehensive and promises to deliver a more thorough understanding of how bacteria can withstand antibiotic treatment under relevant conditions. This approach will lead to the identification of novel antibiotic targets. Finally, this project will validate screens and identify lead compounds for drug discovery projects. This approach will specifically identify compounds that decrease tolerance to conventional antibiotics. The mechanism of action will shed further light on the nature of antibiotic tolerance while also acting as the foundation of an antibiotic development project.